670,000 new cases of leprosy were detected during the year 2002. What are the factors that contributed to this high incidence even though effective multidrug therapy implemented by the World Health Organization has been used to treat leprosy patients over the last 10-20 years? For leprosy, the underlying mechanisms of transmission of Mycobacterium leprae, the causative organism, are still unclear with respect to the source, reservoir, mode of transmission and interaction with the host. Multiple hypotheses exist, but answers remain speculative owing to the inability to cultivate M. leprae in vitro, the slow onset of disease, and the broad spectrum of clinical manifestations. The recent availability of the genome sequence of an isolate of M. leprae is a valuable new resource applicable to the epidemiology of leprosy. This proposal presents a unique approach aimed at a concerted effort to study multiple factors in order to obtain a more comprehensive understanding of the most plausible causes for continued incidence of leprosy. The objectives are to advance the research endeavors at several independent laboratories through development and application of sensitive, specific and simple molecular methods. The specific aims are: 1.To evaluate the contribution of the extra-human environment in leprosy transmission by: a) PCR amplification of M. leprae specific DNA and mRNA in soil and water;and b) strain typing for comparison with clinical strains found in the endemic region;2. To evaluate sub-clinical infections in contacts of leprosy patients by: a) PCR amplification of M. leprae specific DNA and mRNA;b) strain typing for comparison with clinical strains found in index cases;and c) demonstration of mRNA of cytokines involved in infection and immunity;and 3. To evaluate the contribution of incident leprosy cases by: a) PCR amplification of M. leprae specific DNA and mRNA;b) strain typing for comparison with epidemiologically linked cases and;c) identification of primary and acquired drug resistance conferred by mutations in folP and rpoB genes, targets of dapsone and rifampicin respectively. It is important to prove identity of M. leprae within the chain of transmission by strain typing methods. The application of nine newly discovered polymorphic loci for strain typing is a key component of this proposal. It is envisioned that the practical solutions and the knowledge database that will emerge from this research investment will have a tangible impact on leprosy surveillance and control of transmission in the most endemic areas and to studies regarding virulence, evolution and pathogenicity of M. leprae.